Bottom Line:
We confirmed the less aggressive phenotype of BxPC-3 and CAPAN-2 compared with PANC-1 and MIA PaCa-2 cells, through the evaluation of Epithelial-Mesenchymal Transition (EMT) markers.Interestingly, in MIA PaCa-2 cells we found a cleaved form of ANXA1 (33 kDa) that localizes at cellular membranes and is secreted outside the cells, as confirmed by MS analysis.Finally, the treatment of PANC-1 cells with MIA PaCa-2 supernatants significantly increased the migration rate of these cells.

Background: Annexin A1 (ANXA1), a 37 kDa multifunctional protein, is over-expressed in tissues from patients of pancreatic carcinoma (PC) where the protein seems to be associated with malignant transformation and poor prognosis.

Methods: The expression and localization of ANXA1 in MIA PaCa-2, PANC-1, BxPC-3 and CAPAN-2 cells were detected by Western Blotting and Immunofluorescence assay. Expression and activation of Formyl Peptide Receptors (FPRs) were shown through flow cytometry/PCR and FURA assay, respectively. To investigate the role of ANXA1 in PC cell migration and invasion, we performed in vitro wound-healing and matrigel invasion assays.

Results: In all the analyzed PC cell lines, a huge expression and a variable localization of ANXA1 in sub-cellular compartments were observed. We confirmed the less aggressive phenotype of BxPC-3 and CAPAN-2 compared with PANC-1 and MIA PaCa-2 cells, through the evaluation of Epithelial-Mesenchymal Transition (EMT) markers. Then, we tested MIA PaCa-2 and PANC-1 cell migration and invasiveness rate which was inhibited by specific ANXA1 siRNAs. Both the cell lines expressed FPR-1 and -2. Ac2-26, an ANXA1 mimetic peptide, induced intracellular calcium release, consistent with FPR activation, and significantly increased cell migration/invasion rate. Interestingly, in MIA PaCa-2 cells we found a cleaved form of ANXA1 (33 kDa) that localizes at cellular membranes and is secreted outside the cells, as confirmed by MS analysis. The importance of the secreted form of ANXA1 in cellular motility was confirmed by the administration of ANXA1 blocking antibody that inhibited migration and invasion rate in MIA PaCa-2 but not in PANC-1 cells that lack the 33 kDa ANXA1 form and show a lower degree of invasiveness. Finally, the treatment of PANC-1 cells with MIA PaCa-2 supernatants significantly increased the migration rate of these cells.

Conclusion: This study provides new insights on the role of ANXA1 protein in PC progression. Our findings suggest that ANXA1 protein could regulate metastasis by favouring cell migration/invasion intracellularly, as cytoskeleton remodelling factor, and extracellularly like FPR ligand.

Fig2: Effects of ANXA1 knockdown on migration and invasiveness rate of MIA PaCa-2 and PANC-1 cells. Western blot using an anti-ANXA1 antibody on protein extracts from MIA PaCa-2 (panel A) or PANC-1 cells (panel D) treated or not with siRNAs direct against ANXA1 (siANXA1). Δ represents ANXA1 fold change normalized to control levels by densitometry. Protein normalization was performed on tubulin levels. B, Results of wound-healing assay on MIA PaCa-2 (panel B) or PANC-1 cells (panel E) transfected with siANXA1s or scrambled siRNAs. Statistical significance was calculated using unpaired t-test between control and ANXA1 knock-down cells, **p < 0.01 vs untreated control. The data are representative of 5 independent experiments ± SEM. Invasiveness rate of MIA PaCa-2 (panel C) or PANC-1 cells (panel F). In invasion assays a total of 90,000 cells were transfected or not with siANXA1s (5nM) or scrambled siRNAs (5nM) for 72 h and plated as described in Methods section. Invasiveness rate was founded out by counting stained cells on the lower surface of the filters. Data represent mean cell counts of 12 separate fields per well ± SEM of 5 experiments. Statistical significance was calculated using unpaired t-test between control and ANXA1 knock-down cells **p < 0.01 vs serum free (SF) control; ***p < 0.001 vs SF control; ###p < 0.001 vs control.

Mentions:
We observed that in MIA PaCa-2 and PANC-1 cell lines ANXA1 localized in the regions that are involved in the cell movement. As the migration/invasion processes start once cells form actin- and FAK-rich protrusions that adhere to the matrix and create the tension forces necessary for cell motility [30], we hypothesised a role for the protein in these processes. The expression of ANXA1 was greatly reduced in MIA PaCa-2 (Figure 2A) and PANC-1 (Figure 2D) cells by specific siRNA transfection. Thus a wound-healing migration assay on cellular monolayer in ANXA1 knockdown cells was performed. The confluent cultures were scraped to create a wound and cell migration was monitored by time-lapse video-microscopy at the site of the wound. We measured the migration distances of selected cells at different time points as described in Methods section. In ANXA1 knockdown MIA PaCa-2 (Figure 2B) and PANC-1 (Figure 2E) cells the rate of migration decreased in a significant manner, if compared with the wild type control and with scrambled RNA transfected cells.

Fig2: Effects of ANXA1 knockdown on migration and invasiveness rate of MIA PaCa-2 and PANC-1 cells. Western blot using an anti-ANXA1 antibody on protein extracts from MIA PaCa-2 (panel A) or PANC-1 cells (panel D) treated or not with siRNAs direct against ANXA1 (siANXA1). Δ represents ANXA1 fold change normalized to control levels by densitometry. Protein normalization was performed on tubulin levels. B, Results of wound-healing assay on MIA PaCa-2 (panel B) or PANC-1 cells (panel E) transfected with siANXA1s or scrambled siRNAs. Statistical significance was calculated using unpaired t-test between control and ANXA1 knock-down cells, **p < 0.01 vs untreated control. The data are representative of 5 independent experiments ± SEM. Invasiveness rate of MIA PaCa-2 (panel C) or PANC-1 cells (panel F). In invasion assays a total of 90,000 cells were transfected or not with siANXA1s (5nM) or scrambled siRNAs (5nM) for 72 h and plated as described in Methods section. Invasiveness rate was founded out by counting stained cells on the lower surface of the filters. Data represent mean cell counts of 12 separate fields per well ± SEM of 5 experiments. Statistical significance was calculated using unpaired t-test between control and ANXA1 knock-down cells **p < 0.01 vs serum free (SF) control; ***p < 0.001 vs SF control; ###p < 0.001 vs control.

Mentions:
We observed that in MIA PaCa-2 and PANC-1 cell lines ANXA1 localized in the regions that are involved in the cell movement. As the migration/invasion processes start once cells form actin- and FAK-rich protrusions that adhere to the matrix and create the tension forces necessary for cell motility [30], we hypothesised a role for the protein in these processes. The expression of ANXA1 was greatly reduced in MIA PaCa-2 (Figure 2A) and PANC-1 (Figure 2D) cells by specific siRNA transfection. Thus a wound-healing migration assay on cellular monolayer in ANXA1 knockdown cells was performed. The confluent cultures were scraped to create a wound and cell migration was monitored by time-lapse video-microscopy at the site of the wound. We measured the migration distances of selected cells at different time points as described in Methods section. In ANXA1 knockdown MIA PaCa-2 (Figure 2B) and PANC-1 (Figure 2E) cells the rate of migration decreased in a significant manner, if compared with the wild type control and with scrambled RNA transfected cells.

Bottom Line:
We confirmed the less aggressive phenotype of BxPC-3 and CAPAN-2 compared with PANC-1 and MIA PaCa-2 cells, through the evaluation of Epithelial-Mesenchymal Transition (EMT) markers.Interestingly, in MIA PaCa-2 cells we found a cleaved form of ANXA1 (33 kDa) that localizes at cellular membranes and is secreted outside the cells, as confirmed by MS analysis.Finally, the treatment of PANC-1 cells with MIA PaCa-2 supernatants significantly increased the migration rate of these cells.

Background: Annexin A1 (ANXA1), a 37 kDa multifunctional protein, is over-expressed in tissues from patients of pancreatic carcinoma (PC) where the protein seems to be associated with malignant transformation and poor prognosis.

Methods: The expression and localization of ANXA1 in MIA PaCa-2, PANC-1, BxPC-3 and CAPAN-2 cells were detected by Western Blotting and Immunofluorescence assay. Expression and activation of Formyl Peptide Receptors (FPRs) were shown through flow cytometry/PCR and FURA assay, respectively. To investigate the role of ANXA1 in PC cell migration and invasion, we performed in vitro wound-healing and matrigel invasion assays.

Results: In all the analyzed PC cell lines, a huge expression and a variable localization of ANXA1 in sub-cellular compartments were observed. We confirmed the less aggressive phenotype of BxPC-3 and CAPAN-2 compared with PANC-1 and MIA PaCa-2 cells, through the evaluation of Epithelial-Mesenchymal Transition (EMT) markers. Then, we tested MIA PaCa-2 and PANC-1 cell migration and invasiveness rate which was inhibited by specific ANXA1 siRNAs. Both the cell lines expressed FPR-1 and -2. Ac2-26, an ANXA1 mimetic peptide, induced intracellular calcium release, consistent with FPR activation, and significantly increased cell migration/invasion rate. Interestingly, in MIA PaCa-2 cells we found a cleaved form of ANXA1 (33 kDa) that localizes at cellular membranes and is secreted outside the cells, as confirmed by MS analysis. The importance of the secreted form of ANXA1 in cellular motility was confirmed by the administration of ANXA1 blocking antibody that inhibited migration and invasion rate in MIA PaCa-2 but not in PANC-1 cells that lack the 33 kDa ANXA1 form and show a lower degree of invasiveness. Finally, the treatment of PANC-1 cells with MIA PaCa-2 supernatants significantly increased the migration rate of these cells.

Conclusion: This study provides new insights on the role of ANXA1 protein in PC progression. Our findings suggest that ANXA1 protein could regulate metastasis by favouring cell migration/invasion intracellularly, as cytoskeleton remodelling factor, and extracellularly like FPR ligand.